Electrodes, methods, apparatuses comprising micro-electrode arrays

ABSTRACT

Described are micro-arrays of electrodes disposed proximal to a flexible substrate, electronic components and sensors comprising such arrays, and methods of use for such arrays.

FIELD OF THE INVENTION

[0001] The present invention relates to arrays of micro-electrodes, tomethods of preparing the arrays, and to uses of the arrays. The arraysmay be used in conventional applications for electrodes. In oneembodiment of the invention, the array is an interdigitated array, andmay be used as an electrode in an electrochemical sensor.

BACKGROUND

[0002] Electrodes are well known devices which permeate industry, andwhich, although often very small in size and not particularly visible,can have a significant impact on peoples' lives. Electrodes are used inelectronic instruments having many industrial, medical, and analyticalapplications. To name just a few, they include monitoring andcontrolling fluid flow, and various types of analytical methods whereinelectric current is measured to indicate the presence or concentrationof certain chemical species.

[0003] With respect to analytical methods, the need for detection andquantitative analysis of certain chemicals found within a largercomposition can be important for the chemical and manufacturingindustries, as well as biotechnology, environmental protection, andhealth care industries. Examples of substances that may be analyzedinclude liquid samples such as tap water, environmental water, andbodily fluids such as blood, plasma, urine, saliva, interstitial fluid,etc.

[0004] Many analytical techniques, sometimes referred to aselectrochemical detection methods, make use of electrodes as a componentof an electrochemical sensor. The sensors are used in combination withelectronic apparatuses to precisely detect the presence or concentrationof a selected chemical species (analyte) within a substance sample.Techniques that allow the use of miniaturized disposable electroanalyticsample cells for precise micro-aliquote sampling, and self-contained,automatic means for measuring the analysis, can be particularly useful.

[0005] Electrochemical detection methods can include amperometricmeasurement techniques, which generally involve measurement of a currentflowing between electrodes that directly or indirectly contact a sampleof a material containing an analyte, and studying the properties of thecurrent. The magnitude of the current can be compared to the currentproduced by the system with known samples of known composition, e.g., aknown concentration of analyte, and the quantity of analyte within thesample substance can be deduced. These types of electrochemicaldetection methods are commonly used because of their relatively highsensitivity and simplicity.

[0006] Micro-electrode arrays are structures generally having twoelectrodes of very small dimensions, typically with each electrodehaving a common element and electrode elements or micro-electrodes. If“interdigitated” the arrays are arranged in an alternating, finger-likefashion (See, e.g., U.S. Pat. No. 5,670,031). These are a sub-class ofmicro-electrodes in general. Interdigitated arrays of micro-electrodes,or IDAs, can exhibit desired performance characteristics; for example,due to their small dimensions, IDAs can exhibit excellent signal tonoise ratios.

[0007] Interdigitated arrays have been disposed on non-flexiblesubstrates such as silicon or glass substrates, using integrated circuitphotolithography methods. IDAs have been used on non-flexible substratesbecause IDAs have been considered to offer superior performanceproperties when used at very small dimensions, e.g., with featuredimensions in the 1-3 micrometer range. At such small dimensions, thesurface structure of a substrate (e.g., the flatness or roughness)becomes significant in the performance of the IDA. Because non-flexiblesubstrates, especially silicon, can be processed to an exceptionallysmooth, flat, surface, these have been used with IDAs.

SUMMARY OF THE INVENTION

[0008] Whereas micro-electrodes have in the past been used withnon-flexible substrates such as silicon, ceramic, glass, aluminum oxide,polyimide, etc., it has now been discovered that micro-electrode arrays,for example, IDAs, can be advantageously useful when disposed onflexible substrates. Moreover, such micro-electrodes, disposed onflexible surfaces, can be prepared using methods that involve flexiblecircuit photolithography, as opposed to methods relating to integratedcircuit photolithography.

[0009] An interdigitated array of the invention, disposed on a flexiblesubstrate, can be used generally, in applications where IDAs are knownto be usefully employed. In particular embodiments of the invention, theIDAs can be used to construct electrochemical sensors, test cells, ortest strips. The sensors can be used with electronic detection systems(sometimes referred to as “test stands”) in methods of analyzing samplecompositions for analytes. Preferred embodiments of sensors can bedisposable, and can include channels or microchannels, preferably acapillary, which facilitates flow of a substance sample into thereaction chamber and in contact with the sensor.

[0010] The micro-electrode arrays of the invention can be useful whendisposed onto a flexible substrate. In particular, IDAs are shown to beeffective at dimensions relatively larger than the dimensions often usedfor IDAs disposed on non-flexible substrates. Even though they can berelatively larger than IDAs disposed on non-flexible substrates, theinventive IDAs are still able to exhibit performance properties, e.g.,signal to noise amplification benefits and steady-state assay profiles,comparable to IDAs having smaller dimensions.

[0011] Electrochemical sensors of the invention have been found toprovide performance advantages, e.g., relative to commercially availablesensors. For sensors used in glucose monitoring, compared tocommercially available sensors, the inventive sensors can exhibitimproved (shortened) processing periods, e.g., one half second tosteady-state after application of the assay potential and 5 seconds toreadout, and the ability to get an accurate and precise readout from arelatively small sample of substance, e.g., less than one microliter(μl), preferably a sample volume in the range from about 0.25 to 0.1 μl,e.g., from about 0.4 to about 0.1 μl.

[0012] The use of larger-dimensioned micro-electrode arrays also allowsthe significant advantage of fabricating arrays and sensors usingrelatively less expensive and more efficient flex circuitphotolithography processes. These can advantageously incorporate the useof solid materials instead of spin-on liquid materials, e.g., one ormore of a solid photoresist or a solid coverlay, instead of liquidmaterials typically used in integrated circuit photolithography.

[0013] An aspect of the invention relates to micro-electrodes used incombination with a flexible substrate. The array can include a workingelectrode and a counter electrode, each including a common lead andcommonly-connected electrode elements, for example with the electrodeelements being arranged in a substantially-parallel, alternatingfashion. Preferred dimensions for micro-electrodes can be, e.g., featuresize or width of electrodes (W_(e)) in the range from 15 or 20 or 25 μm,up to about 100 μm, more preferably from greater than or about 25 or 30μm to about 50 μm. Preferred spacing between electrodes (W_(g)) can alsobe in the range from about 15 to about 50 μm, more preferably fromgreater than or about 20 or 25 μm to about 45 μm.

[0014] Another aspect of the invention relates to an electrochemicalsensor comprising an array of micro-electrodes disposed on a flexiblesubstrate. The sensor can further include a chemical coating disposed onthe array to facilitate practice of electrochemical detection methods.

[0015] Yet another aspect of the invention relates to a method ofdetecting an analyte using an array of micro-electrodes of theinvention, e.g., using an electrochemical sensor comprising aninterdigitated array disposed proximal to a flexible substrate. Such amethod can include certain of the following steps. A sensor is providedwhich comprises micro-electrodes proximal to a flexible substrate, and achemical coating proximal to the micro-electrodes; the coating comprisesa compound reactive to produce an electroactive reaction product. Thecoating is contacted with a substance comprising an analyte, allowingthe analyte to react with chemical components of the coating to producean electroactive reaction product. Electric properties of the coatingcan be measured, and the electric properties can be correlated to theamount of electroactive reaction product, and to the amount of analyte.

[0016] Still another aspect of the invention relates to a method ofpreparing a micro-electrode, including the step of disposing themicro-electrode onto a flexible substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows an embodiment of an interdigitated array of theinvention.

[0018]FIG. 2 shows a top view of a sensor of the invention.

[0019]FIG. 3 shows a top view of a sensor of the invention.

[0020]FIG. 4 shows a side view of a sensor of the invention.

[0021]FIG. 5 shows a side view of a sensor of the invention.

[0022]FIG. 6 shows a perspective view of a dissembled sensor of theinvention.

[0023]FIG. 7 shows data of assay current versus blood glucose level.

DETAILED DESCRIPTION

[0024] An embodiment of the present invention is directed to arrays ofmicro-electrodes, e.g., an interdigitated array of electrodes (sometimesreferred to as “microband” electrodes) used in combination with aflexible substrate.

[0025] An array of micro-electrodes includes two electrodes, referred toas the working electrode and the counter electrode, electricallyinsulated from one another.

[0026] Micro-electrodes, as distinguished from other electrodesgenerally, are understood in the electronic and biosensor arts. Inanalyzing a liquid sample using electrodes and electronic equipment andtechniques, the size and spacing of electrodes can affect whetherdiffusion of an analyte through the sample to an electrode occurs by aplanar or non-planar path. Micro-electrode arrays are of a size andspacing such that in detecting chemical species of a solution, thespecies will diffuse toward or approach an electrode of themicro-electrode array in a non-planar fashion, e.g., in a curved orhemispherical path of diffusion. In contrast, non-microelectrodes, i.e.,“macro-electrodes,” cause diffusion of an analyte through a soluteaccording to a substantially planar path. It is also understood thatsome electrode configurations can cause diffusion to take place by a mixof planar and non-planar paths, in which case the electrodes can beconsidered a micro-electrode array, especially if the diffusion occurspredominantly (e.g., greater than 50%) according to a non-planar path,or if the size of the electrodes is less than 100 μm, e.g., less than 50μm.

[0027] The electrodes of a micro-electrode array are positioned neareach other in an arrangement that will result in non-planar diffusion asdescribed. The arrangement of the electrodes can be any arrangement thatresults in such diffusion, with a working and a counter electrode beingsubstantially evenly spaced from each other. One electrode may bearranged into a shape or figure or outline that will produce intersticeswithin which the second electrode may be placed. For instance, oneelectrode,can be arranged as an increasing radius, substantiallycircular spiral, with a continuous, long and narrow interstitial areabeing created between each successively larger revolution of electrode.The other electrode can be positioned in the interstitial area betweenrevolutions, while the electrodes remain insulated from one another. Thewidth and spacing of the electrodes can be arranged to result inmicro-electrode array performance.

[0028] According to other forms of such micro-electrode arrays, thespiral may not be substantially circular, but could include linear,square, angled, or oblong or oval features. Or, the electrodes could bearranged in any other geometric form whereby the electrodes are placedadjacent to each other and within the other's respective interstitialarea, e.g., by following a similar path separated by a substantiallyuniform gap.

[0029] In one particular embodiment, the micro-electrode can be arrangedinto an interdigitated array, meaning that at least a portion ofelectrode elements of the working electrode are placed substantiallyparallel to and in alternating succession with at least a portion of theelectrode elements of the counter electrode, e.g., in an alternating,“finger-like” pattern. Such interdigitated micro-electrode arraysinclude electrode elements (sometimes referred to as “fingers”) and acommon element (“contact strip”) which commonly connects the electrodeelements.

[0030] The components of the electrodes may be made of any conductivematerial, including those known and conventionally used as electrodematerials, particularly including materials known in the flexiblecircuit and photolithography arts. These can include, for example,carbon, noble metals such as: gold, platinum, palladium, alloys of thesemetals, potential-forming (conductive) metal oxides and metal salts, aswell as others.

[0031] The electrodes and their components can be of dimensions, meaningthe width of the electrode components as well as the separation betweencomponents, that can provide an array with useful properties, e.g.,useful or advantageous capabilities with respect to contacting asubstance or measuring electrical properties. Advantageously,interdigitated arrays can be prepared at dimensions that allow forcontact with and measurement of electrical properties of a relativelysmall sample of a substance.

[0032] In preferred embodiments of the invention, each electrode elementcan independently have a width (W_(e)) in the range from greater than 15micrometers (μm) to about 50 μm, with the range from greater than orabout 20 or 25 μm to about 40 μm being particularly preferred. Theseparation between electrode components (W_(g)), especially theseparation between alternating electrode elements, can also preferablybe in the range between about 15 micrometers and about 50 μm, with therange from greater than or about 20 or 25 μm to about 40 μm beingparticularly preferred. The total area of an electrode (meaning the areaof the fingers but not the common element) can be chosen depending onthese dimensions, on the use intended for the electrode, on the desiredcurrent level intended to pass through the electrode, and on the desirednumber of electrode elements. An exemplary area of an electrode having10 electrode elements can be in the range from about 0.1 to about 0.5square millimeters, (for example 10 electrode fingers having dimensionsof 50 μm by 1 mm), e.g., from about 0.2 to 0.3.

[0033] The thickness of the electrode components can be sufficient tosupport a desired electric current. Exemplary thicknesses can be in therange from about 30 to 200 nanometers (nm), with a preferred thicknessbeing about 100 nm.

[0034] The electrodes can independently have a number of interdigitatedelectrode elements sufficient to provide utility, e.g., allowing contactwith a substance to measure its electrical behavior. Conventionally, thearray can have substantially the same number (equal, plus or minus one)of electrode elements in the working electrode as are in the counterelectrode, allowing the electrode elements to be paired next to eachother in an alternating sequence. In some preferred embodiments of thearray, such as in some of the applications described below forelectrochemical sensors, each electrode of an array may typically havefrom about 4 to about 30 electrode elements.

[0035]FIG. 1 illustrates an embodiment of an array of the invention.Working electrode 2 and counter electrode 4 are arranged as aninterdigitated array on flexible substrate 10. (The figure is not toscale and its dimensions, as well as the dimensions of the otherfigures, should not be construed to limit the invention). The workingand counter electrodes include common strips 6 a and 6 b, respectively,which can be connected to electrically conductive means (e.g.,“connectors,” “pads,” or “leads,” etc.) for connecting the electrodes toan external circuit. In the illustrated example, the working electrodeincludes electrode elements 8 a connected to common strip 6 a, and thecounter electrode includes electrode elements 8 b connected to commonstrip 6 b.

[0036] According to the invention, the interdigitated array is disposedproximal to, e.g., on, a flexible substrate. To act as a flexiblesubstrate, a material must be flexible and also insulating, and istypically relatively thin. The substrate should be capable of adheringcomponents of an IDA, or additional components of a sensor, to itssurface. Such thin, insulative, flexible substrates are known in the artof flexible circuits and flex circuit photolithography. “Flexiblesubstrates” according to the present disclosure can be contrasted tonon-flexible substrates used in integrated circuit (IC) photolithographybut not in flexible circuit photolithography. Examples of non-flexiblesubstrates used in IC photolithography include silicon, aluminum oxide,and other ceramics. These non-flexible substrates are chosen to beprocessable to a very flat surface. Typical flexible substrates for usein the invention are constructed of thin plastic materials, e.g.,polyester, especially high temperature polyester materials; polyethylenenaphthalate (PEN); and polyimide, or mixtures of two or more of these.Polyimides are available commercially, for example under the trade nameKapton®, from I. E. duPont de Nemours and Company of Wilmington, Del.(duPont). Polyethylene naphthalate is commercially available asKaladex®, also from duPont. A particularly preferred flexible substrateis 7 mil thick Kaladex® film.

[0037] Interdigitated arrays of the invention can be used inapplications generally known to incorporate electrodes, especiallyapplications known to involve interdigitated arrays of electrodes.Various applications are known in the arts of electronics andelectrochemistry, including applications relating to process and flowmonitoring or control, and chemical analytical methods. The arrays maybe particularly useful as a component of an electrochemical sensor,where there is added value, benefit, or cost efficiency, to the use of aflexible substrate, or where there is value, benefit, or cost efficiencyin having an interdigitated array of dimensions relatively larger thanthe dimensions of interdigitated arrays conventionally disposed onnon-flexible substrates.

[0038] An interdigitated array of the invention can, for example, beincluded in an electrochemical sensor (sometimes referred to as a“biosensor” or simply “sensor”) used in electrochemical detectionmethods. Electrochemical detection methods operate on principles ofelectricity and chemistry, or electrochemistry, e.g., on principles ofrelating the magnitude of a current flowing through a substance, theresistance of a substance, or a voltage across the substance given aknown current, to the presence of a chemical species within thesubstance. Some of these methods can be referred to as potentiometric,chronoamperometric, or impedence, depending on how they are practiced,e.g., whether potential difference or electric current is controlled ormeasured. The methods and sensors, including sensors of the invention,can measure current flowing through a substance due directly orindirectly to the presence of a particular chemical compound (e.g., ananalyte or an electroactive compound), such as a compound within blood,serum, interstitial fluid, or another bodily fluid, e.g., to identifylevels of glucose, blood urea, nitrogen, cholesterol, lactate, and thelike. Adaptations of some electrochemical methods and electrochemicalsensors, and features of their construction, electronics, andelectrochemical operations, are described, for example, in U.S. Pat.Nos. 5,698,083, 5,670,031, 5,128,015, and 4,999,582, each of which isincorporated herein by reference.

[0039] Oftentimes, a compound of interest (analyte) in a substance isnot detected directly but indirectly, by first reacting the analyte withanother chemical or set of chemicals proximal to or in contact with anIDA. The reaction produces an electroactive reaction product that iselectrochemically detectable and quantifiable by applying a potentialdifference between the counter and working electrodes and measuring themagnitude of the current produced. This allows measurement of the amountof electroactive reaction product generated by the first reaction, andcorrelation of that measurement to the amount of analyte in the samplesubstance.

[0040] An example of such a method involves the catalytic use of anenzyme, and is sometimes referred to as enzymatic amperometry. Thesemethods can use an interdigitated array of electrodes coated with achemical coating that contains a chemical compound reactive to producean electroactive reaction product. (The chemical compound reactive toproduce an electroactive reaction product is sometimes referred toherein as a “mediator.”) Upon contacting the coating with a sample thatcontains an analyte, analyte reacts with chemical compounds of thecoating to generate electroactive reaction product. This electroactivereaction product can be electronically detected, measured, orquantified, by applying a potential difference between the electrodesand measuring the current generated by the electrooxidation of themediator at the working electrode. By calibrating the system's behaviorusing known substances and concentrations, the electrical behavior ofthe system in the presence of a sample substance of unknown compositioncan be determined by comparison to the calibration data.

[0041] The sensor of the invention may be used in amperometricapplications, e.g., enzymatic amperometric applications, if disposed onthe array is a coating of useful chemistry, including e.g., an enzymeand a mediator. When a sample containing an analyte is contracted withthe coating, the analyte, enzyme, and the mediator participate in areaction, wherein the mediator is either reduced (receives at least oneelectron) or is oxidized (donates at least one electron). Usually, inthis reaction, the analyte is oxidized and the mediator is reduced.After this reaction is complete, an electrical potential difference canbe applied between the electrodes. The amount of reducible species andthe applied potential difference must be sufficient to causediffusion-limited electrooxidation of the reduced form of the mediatorat the surface of the working electrode. The IDA electrode configurationof the sensor places the working electrode fingers in close proximity tocounter electrode fingers. Mediator electrooxidized at the workingelectrode can therefore diffuse rapidly to the adjacent counterelectrode via radial diffusion where it is once again reduced. Likewise,oxidized mediator reduced at the counter electrode can migrate to theworking electrode for electrooxidation to the oxidized form. Thismigration between the fingers produces a constant or “steady state”current between the electrodes. After a short time delay, this steadystate current is measured and correlated to the amount of analyte in thesample.

[0042] The chemistries of the first and second reactions can be of anynature effective to produce the electroactive reaction product of thefirst reaction,/to detect or quantify the electroactive reaction productduring the second reaction, and to allow correlation of the amount ofelectroactive reaction product with the presence or concentration ofanalyte in the original sample.

[0043] In general, a typical first reaction can be anoxidation/reduction sequence, preferably occurring without the need fora chemical potential across the electrodes. It can be desirable for thisreaction to favor maximum, preferably complete conversion of theanalyte, and to proceed as quickly as possible. Often this reaction iscatalyzed, e.g., enzymatically. Such reaction schemes and theirapplication to enzymatic amperometry are known. See, e.g., U.S. Pat. No.5,128,015; European Patent Specification EP 0 406 304 B1; and Aoki,Koichi, Quantitative Analysis of Reversible Diffusion-ControlledCurrents of Redox Soluble Species at Interdigitated Array ElectrodesUnder Steady-State Conditions, J. Electroanal. Chem. 256 (1988) 269-282.An example of a useful reaction scheme can be the reaction of acomponent of a bodily fluid, e.g., glucose, with an enzyme and acofactor, in the presence of a mediator, e.g., an oxidizer, to producean electroactive reaction product.

[0044] The chemistry of a first reaction scheme of any chosenelectrochemical detection method can be chosen in light of variouschemical factors relating to the system, including the identity of theanalyte and of the sample substance. Even then, for a given analyte orsubstance, various different reactive components may be useful in termsof a catalyst (often, a variety of enzymes will be useful), co-reactants(e.g., a variety of mediators may be useful), and cofactors (if needed,a variety may be useful). Many such reaction schemes and their reactivecomponents and reaction products are known, and examples of a fewdifferent enzymes include those listed in Table 1. Redox MediatorAdditional Analyte Enzymes (Oxidized Form) Mediator Glucose GlucoseFerricyanide, dehydrogenase osmium (III)- and Diaphorase (bipyridyl)-2-imidazolyl-chloride, Meldola blue, [Ru(NH₃)₅MeIm] Cl3 [OS(III)(NH₃)₅pyz]₂(SO₄)₃, NITROSO analine derivatives Glucose Glucose oxidase(see above) Cholesterol Cholesterol (see glucose) 2,6-Dimethyl-1,esterase and 4-Benzoquinone, Cholesterol 2,5-Dichloro-1, oxidase4-benzoquinone, or phenazine ethosulfate HDL Cholesterol (see glucose)2,6-Dimethyl-1, Cholesterol esterease and 4-Benzoquinone, Cholesterol2,5-Dichloro-1, oxidase 4-benzoquinone, or phenazine ethosulfateTriglycerides Lipoprotein (see glucose) Phenazine lipase, Glycerolmethosultate, kinase, phenazine Glycerol-3- ethosulfate. phosphateoxidase Triglycerides Lipoprotein (see glucose) Phenazine lipase,Glycerol methosultate, kinase, phenazine Glycerol-3- ethosulfate.phosphate dehydrogenase and Diaphorase Lactate Lactate oxidase (seeglucose) 2,5-Dichloro-1, 4-benzoquinone Lactate Lactate (see glucose)dehydrogenase and Diaphorase Lactate Diaphorase (see glucose) Dehydro-genase Pyruvate Pyruvate (see glucose) oxidase Alcohol Alcohol oxidase(see glucose) Alcohol Alcohol (see glucose) dehydrogenase and DiaphoraseUric acid Uricase (see glucose) 3-Hydroxy- 3- (see glucose) butric acidHydroxybutyrate (ketone dehydrogenase bodies) and Diaphorase

[0045] A mediator can be any chemical species (generally electroactive),which can participate in a reaction scheme involving an enzyme, ananalyte, and optionally a cofactor (and reaction products thereof), toproduce a detectable electroactive reaction product. Typically,participation of the mediator in this reaction involves a change in itsoxidation state (e.g., a reduction), upon interaction with any one ofthe analyte, the enzyme, or a cofactor, or a species that is a reactionproduct of one of these (e.g., a cofactor reacted to a differentoxidation state). A variety of mediators exhibit suitableelectrochemical behavior. A mediator can preferably also be stable inits oxidized form; may optionally exhibit reversible redoxelectrochemistry; can preferably exhibit good solubility in aqueoussolutions; and preferably reacts rapidly to produce an electroactivereaction product. Examples of suitable mediators include benzoquinone,medula blue, other transition metal complexes, potassium ferricyanide,and nitrosoanalines, see U.S. Pat. No. 5,286,362. See also Table 1.

[0046] To describe an example of an oxidation/reduction reaction schemethat is known to be useful for detecting glucose in human blood, asample containing glucose can react with an enzyme (e.g.,Glucose-Dye-Oxidoreductase (Gluc-Dor)) and optionally a cofactor, (e.g.,pyrrolo-quinoline-quinone), in the presence a redox mediator (e.g.,benzoquinone, ferricyanide, or nitrosoanaline derivatives), to producethe oxidized form of the analyte, gluconolactone, and the reduced formof the redox mediator. See U.S. Pat. No. 5,128,015. Other examples ofreaction schemes are known, and are typically used in methods designedto detect a specific analyte, e.g., cholesterol, urea, etc.

[0047] After the reaction is complete, a power source (e.g., battery)applies a potential difference between the electrodes. When thepotential difference is applied, the amount of oxidized form of theredox mediator at the counter electrode and the potential differencemust be sufficient to cause diffusion-limited electrooxidation of thereduced form of the redox mediator at the working electrode surface. Inthis embodiment, the close proximity of the counter and workingelectrode fingers in the IDA electrode configuration aids in the fastradial diffusion of the reduced and oxidized redox mediator between theelectrodes. Recycling of the mediator between the electrodes and theirsubsequent oxidation and reduction on the electrodes generates aconstant or “steady state” assay current. This steady state assaycurrent is measured by a current measuring meter.

[0048] The measured current may be accurately correlated to theconcentration of analyte in the sample when the following requirementsare satisfied:

[0049] 1) the rate of oxidation of the reduced form of the redoxmediator is governed by the rate of diffusion of the reduced form of theredox mediator to the surface of the working electrode; and

[0050] 2) the current produced is limited by the oxidation of thereduced form of the redox mediator at the surface of the workingelectrode.

[0051] In the preferred embodiment, these requirements are satisfied byemploying a readily reversible mediator and by using a mixture ofamounts of mediator and other components of the chemical layer to ensurethat the current produced during diffusion limited electrooxidation islimited by the oxidation of the reduced form of the mediator at theworking electrode surface. For current produced during electrooxidationto be limited by the oxidation of the reduced form of the mediator atthe working electrode surface, the amount of reducible species at thesurface of the counter electrode must always exceed the amount of thereduced form of the redox mediator at the surface of the workingelectrode.

[0052] An example of a reaction scheme relates to the detection ofglucose using ferricyanide and Glucose-Dye-Oxidoreductase (Glur-Dor).The electroactive reaction product of the enzymatic reaction betweenglucose and the enzyme is the reduced mediator, ferrocyanide. Theferrocyanide is electrooxidized at the working electrode back toferricyanide. One mole of oxidized redox mediator is reduced at thecounter electrode for every mole of reduced redox mediator oxidized atthe working electrode. Ferricyanide electrooxideized at the workingelectrode, diffuses to the counter electrode, and the ferrocyanideproduced at the counter electrode can rapidly diffuse to the workingelectrode where it is again oxidized. A “quasi-steady state”concentration gradient is established between the counter and workingelectrode pairs resulting in generation of a constant quasi-steady statecurrent at the working electrode.

[0053] The magnitude of the current, preferably as measured at aquasi-steady-state condition, can be correlated to the amount ofelectroactive reaction product present in the coating, and consequently,to the amount of analyte in the sample.

[0054] The chemical coating should allow diffusion of analyte into thecoating, followed by reactions as described. The coating can includematerials which can contain the reactive chemical components, whichallow reaction between the components to product an electroactivereaction product, which allow necessary diffusion of chemicalcomponents, and which can support a current passing through the coatingbased on the concentration of electroactive reaction product. Typically,the coating can be made up of a binder that contains a set of chemicalswhich react to produce an electroactive reaction product. The chemicalsgenerally include a mediator and necessary enzymes and cofactors. Such acoating can also contain a variety of additional components to make thecoating operative and suitable for processing, including specificcomponents listed above as well as surfactants, film formers, adhesiveagents, thickeners, detergents, and other ingredients and additives thatwill be understood by an artisan skilled in the electrochemical sensorart.

[0055] The binder can provide integrity of the coating while allowingdiffusion of the different components of the reaction scheme, reactionbetween the reactive components, and movement of reactive components andproducts sufficient to produce a quasi-steady-state concentrationgradient of mediator and electroactive reaction product and therebyestablish a stable or quasi-steady-state current between the electrodepairs. Exemplary binders can include gelatin, carrageenan,methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, alignate,polyethylene oxide, etc.

[0056] A sensor according to the invention can be understood to includea micro-electrode disposed on a flexible substrate, optionally includinga chemical coating, and further including any immediate appurtenancenecessary to use the sensor in an electronic system or apparatus (e.g.,test stand) designed, for example, for use in an electrochemicaldetection method. A sensor can include the interdigitated array disposedon a flexible substrate, with additional components to independentlyconnect each of the separate electrodes to a different voltage, e.g.,electrical connectors, leads, or pads. In some circumstances, the sensormay include a reference electrode provided on the same or a differentsubstrate and electrically insulated from the interdigitated array. Thesensor may also include components to direct flow of a sample substanceinto contact with the IDA, e.g., a vessel, channel, microchannel, orcapillary. A particularly preferred embodiment of the sensor includes amicrochannel or capillary, most preferably a capillary, which directsflow of a sample substance into the reaction chamber and over the IDA(e.g., a coated IDA).

[0057] A capillary can be included in a sensor to facilitate analysis ofa small volume of a sample substance by precisely directing the flow ofa volume of sample over the IDA, preferably in a short period of time.Analysis of relatively small volumes of a sample substance can beaccomplished, at least in part, due to the signal amplification featuresof the IDA.

[0058] Preferred dimensions of a capillary for what can be referred toas a “low volume sensor configuration,” can be in the range of 0.025 mmto 0.2 mm (depth), preferably about 0.125 mm (depth),×1 mm (width)×3 mm(length), resulting in a capillary chamber requiring a relatively smallvolume of sample, e.g., less than 400 nanoliters (nL). The volume of thechamber can preferably be such that a low volume sample of a substancecan be directed into or through the chamber for analysis. Chambervolumes will vary depending on the type of analyte being studied, andeven its concentration of an analyte. (Blood samples of differenthematocrits will dispense differently into a capillary.) Exemplarychamber volumes can be in the range from about 100 to 300 nanoliters forglucose analysis in interstitial fluid, and from about 250 to 400microliters for glucose analysis applications in the whole blood. In themost preferred embodiments of the sensor, including a capillary, thecapillary may have a vent to facilitate flow of a sample substance intothe capillary chamber by equalizing pressure between the interior andexterior of the chamber.

[0059] The sensor of the invention can include these and other features,and, especially if an embodiment is disposable, can be referred to as a“test strip” or a “test cell.” The term “disposable” refers to sensorsdesigned or sold for a single use, after which they are to be discardedor otherwise stored for later disposal.

[0060] Capillaries may be fabricated as a component of a sensor, usingphotolithographic methods, e.g., as described infra.

[0061] An example of a sensor construction is shown in FIG. 2, accordingto the preferred embodiment. The figure shows sensor 20, including aninterdigitated array of electrodes 22 disposed on flexible substrate 24.The electrodes are connected to electrically-conductive connectors 26which include portions 28 that can be identified as pads, located on thesurface of the flexible substrate, where they are available to becontacted to an external electronic circuit such as a testing apparatus.The connectors also include connector portions 30, which connectelectrode elements at the array to the pads, and which may typically becovered by an insulating layer. FIG. 2a shows a close-up of array 22,showing that electrodes attached to each of connectors 26 are arrangedin an inter digitated fashion (as shown in FIG. 1).

[0062]FIG. 3 shows different details of a sensor of the invention. FIG.3 shows sensor 20 comprising flexible substrate 24, an array ofinterdigitated electrodes 22, and connectors and pads. Non-conductivelayer 32 is disposed over the substrate and connector portions 30 of theconnectors 26, over portions of the array 22, and not over a rectangularcapillary portion including some of the substrate and an intersection ofarray 22; this rectangular portion defines capillary chamber 34. (Achemical coating, not shown in this figure, is preferably disposed overthe array, within the capillary chamber.) Foil 36 covers a rectangularportion of the sensor, including portions of the non-conductive layer32, and a portion of capillary chamber 34, except for air vent 38. Thisembodiment is shown from one side in FIG. 4, and from another side inFIG. 5. FIG. 5 specifically illustrates substrate 24, array 22,non-conductive layer 32, which defines chamber 34, and foil 36. FIG. 5additionally includes coating 40 disposed on array 22, within thecapillary.

[0063]FIG. 6 illustrates an exploded view of a sensor of the invention.The sensor 20 includes flexible substrate 24; a conductive film 40patterned with an interdigitated array of electrodes 22 and connectors26 which include pad portions 28 and connecting portions 30, aninsulating material 32 which defines the depth and dimensions ofcapillary chamber 34, a chemical coating 40 disposed in the capillarychamber 34, and top foil 36 coated with a hydrophilic adhesive layer 42.

[0064] The array of the invention, in various embodiments such as asensor, can be used in electrochemical detection methods, includingthose using the principles and specific methods described above, andothers. Such methods employ the array disposed on a flexible substrate,preferably further including a chemical coating contacting the array.

[0065] Upon contacting the coating with a sample containing analyte,analyte generally diffuses into the coating at a rate dependant onfactors such as the chemical composition of the coating and the chemicalidentity of the analyte. Generally, the chemical coating will be atleast partly solubilized or hydrated by the sample substance. For amethod to provide the quickest read time (the time following contactwith a substance sample, when a reading of the concentration of analytein the substance is available), it is desirable that the analyte diffusequickly into the coating, and thereafter quickly and completely react toproduce an electroactive reaction product. The period during which thisoccurs can be reduced by operating on a relatively small volume ofsample, and by using a sensor having a relatively small amount ofchemical coating to be solubilized or hydrated.

[0066] The time from when the substance containing the analyte iscontacted with the chemical coating until an assay potential is appliedto the array, and during which the analyte diffuses into the coating andreacts to produce an electroactive reaction product, can be referred toas the “delay period”. This period can be any amount of time necessaryfor the above occurrences to transpire, is preferably minimized, and insome embodiments can be less than about 10 seconds, preferably in therange from about 2 to 6 seconds.

[0067] After a delay period, the electric properties of the coating canbe measured. By chronoamperometric methods, or by potentiometricmethods, as will be appreciated by the skilled artisan, either thecurrent or the applied potential can be controlled, and any of therelated current, resistance, or voltage can be measured and correlatedto amounts of electroactive reaction product and analyte. The magnitudeof the current, or alternatively potential difference or the resistanceof the chemical coating, can be measured using an external circuitconnected to the sensor electrodes.

[0068] As an example, according to chronoamperometric methods, apotential (“assay potential”) can be applied across the electrodes,inducing a current (“assay current”) to flow through the coating. Thepotential should be enough to cause reduction or oxidation of the redoxproducts formed in the first step of a binary reaction scheme (e.g., asdescribed above), but should not be sufficient to cause otherelectrochemical reactions or to otherwise cause significant current toflow through the coating. The assay potential can be chosen depending onthe redox mediator chosen, factors relating to the electrochemicaldetection method, the electrochemical system and reaction scheme, andthe general capabilities of the sensor. A typical potential can be inthe range of a few to several hundred millivolts, e.g., from about 100to 500, preferably 200 to 400 millivolts.

[0069] A measured current can initially exhibit a spike to a relativelyelevated level, and can then descend to a steady-state current based ona quasi-steady-state concentration gradients and a recycle reaction loopof the mediator and electroactive reaction product. Preferably, themagnitude of the current can be measured at a time when current flowingthrough this system has approached a plateau, based onquasi-steady-state concentration gradients. The period of time startingwith application of the assay potential and lasting to the plateau ornear-steady-state current can be referred to as the “assay period.”Steady-state assay currents can occur within various such time periods,depending upon the reaction scheme, the chemistries of its components,etc. In the practice of the invention, assay periods of less than oneminute are preferred, e.g., less than 30 seconds, and assay periods ofeven shorter duration, less than 10 seconds, are most preferred. Theassay profile (the profile of the assay current over time) can be tosome extent controlled by changing the spacing between electrodeelements in the array; increased spacing between electrode elements canresult in a longer time interval between assay potential application andformation of the steady state assay currents.

[0070] Assay currents exhibited by exemplary sensors of the inventioncan be any current that will function in an electrochemical detectionmethod. For the sensors of the invention, any useful current can beused, preferably with a range between a lower end in the nanoamp range(e.g., between 20 to 25 nanoamps) up to the microamp range e.g., 100microamps, being an exemplary working range, e.g., at the steady statecurrent plateau. Typical steady state assay currents can be in the rangefrom below one microamp up to around 100 microamp, preferably from about0.5 to about 25 microamps. In an embodiment of the invention useful fordetecting glucose content of a blood sample, the current response(steady state assay current) in this range has been found to be linearwith respect to the concentration of glucose in the sample, particularlyfor glucose concentrations in the range from about 0 to 600 milligramsper deciliter (mg/dL).

[0071] Sensors of the invention may be used in cooperation withelectronic or computerized systems and apparatuses, and in combinationwith methods for identifying analytes and measuring concentration ofanalytes within a substance sample. For example, a sensor can be usedwith a VXI or Biopotentiostat test stand built from components purchasedfrom National Instrument Corp., Austin, Tex. In this context, the methodof the invention can be practiced with a delay period of around 3seconds, an assay potential of about 300 millivolts, and an assay periodwhich, although variable, can preferably be in the range from about 1.5to 2 seconds after applying the assay potential.

[0072] The sensors can be used in such a method to detect and quantifythe concentration of an analyte within a sample substance. The analytecan be chosen from various chemical compounds present within any of alarge variety of substances, generally fluids. Examples of analytesinclude glucose, cholesterol, urea, and the like. Examples of substancescontaining the analyte include bodily fluids such as blood, urine, andinterstitial fluid; water such as environmental water, ground water,waste water, etc.

[0073] In some embodiments of the invention, analytes can be detected atvery low concentrations, for example glucose can be measured atcocentrations as low as 0.5 mg/dL (5 ppm) in blood using ferricyanide asthe mediator.

[0074] The use of an array or sensor of the invention offers certainpractical advantages. For instance, a flexible substrate can be used incombination with relatively larger-dimensioned electrodes, includingelectrode components of increased size (e.g., width) as well asincreased spacing between them. Lower sample volumes can independentlydecrease the time of the delay period. A shorter delay period incombination with an expedited formation of a quasi-steady-state regionof the assay current produces a quicker read time. In the practice ofthe invention, read times of less than 10 seconds have been achieved,with a read times in the range from about 4 and 5 seconds beingpreferred.

[0075] Test cells and test strips according to the invention allow forcontrolled volumes of blood to be analyzed without pre-measuring.Insertion of the test cell into an electronic or computer-controlledapparatus (referred to generally as a test stand) permits automaticfunctioning and timing of the reaction and analysis of the sample. Thisallows for patient self-testing with a very high degree of precision andaccuracy. The method, the sensor or test cell, and the apparatus, aredesigned to provide self-monitoring by a patient of important bodilyfluids, e.g., blood glucose levels. The sensor is used to control thesample volume and reaction media, to provide precise, accurate, andreproducible analysis. Preferably, disposable test strips or test cellscan be used in combination with a portable electrochemical testingmeter.

[0076] The preferred embodiment of the present invention uses amicro-electrode array consisting of interdigitated micro-band electrodesas described above. Although this arrangement leads to theaforementioned re-cycling of redox products between narrowly separatedworking and counter electrodes, this is not a strict requirement forsuccessful practice of the invention. An alternative embodiment is theprovision of an array of more general micro-electrodes to act as theworking electrode structure. These may be micro-bands that are notinterdigitated with the counter electrode, or micro-disks, also notclosely spaced with the counter electrode. In this case the width ordiameter of the working electrode bands or disks should be of such adimension as to allow for some degree of radial or spherical diffusionto the working electrode surfaces. Typically, this dimension should bein the range of 5 to 50 μm, and most preferably 10 to 50 μm for the caseof aqueous systems such as encountered with a sensor used for the assayof biological fluids. In both cases the counter electrode is provided ata distance from the working array that is generally larger than thesmallest dimension of the working electrodes.

[0077] In these embodiments, specific recycling of redox species betweenthe working and counter electrodes is not observed in the same way as inother described embodiments, and assay current magnitudes areconsequently reduced. Nevertheless, the effect of radial or sphericaldiffusion to working micro-electrode structures can still be observed ascurrent densities that are greater than that predicted from lineardiffusion alone. Although reduced in magnitude, and not approachingquasi-steady-state as displayed by the preferred embodiments, it isstill possible to measure dose responses to the analyte in question(e.g. glucose) when the same reagent as described above is disposed onthe micro-electrode array.

[0078] Micro-electrode arrays of the invention can be disposed onto aflexible substrate using various methods useful for disposing electroniccomponents onto substrates, especially flexible substrates. A variety ofsuch methods are generally known for fabrication of different types ofcircuitry, and include specific techniques of dry-coating, lamination,spin-coating, etching, and laser ablation. One or more of the followinggeneralized methods may be specifically useful to prepare microelectrodearrays according to the invention.

[0079] One method of preparing a micro-electrode array as describedherein, e.g., an IDA, is by the use of laser ablation techniques.Examples of the use of these techniques in preparing electrodes forbiosensors are described in U.S. patent application Ser. No. 09/866,030,“Biosensors with Laser Ablation Electrodes with a Continuous CoverlayChannel” filed May 25, 2001, and in U.S. patent application Ser. No.09/411,940, entitled “Laser Defined Features for Patterned Laminates andElectrode,” filed Oct. 4, 1999, both disclosures incorporated herein byreference.

[0080] In general, laser ablative techniques use a laser to cut or molda material. According to the invention, micro-electrodes can be preparedusing ablative techniques, e.g., by ablating a multi-layer compositionthat includes an insulating material and a conductive material, e.g., ametallic laminate of a metal layer coated on or laminated to aninsulating material. The metallic layer may contain pure metals oralloys, or other materials which are metallic conductors. Examplesinclude aluminum, carbon (such as graphite), cobalt, copper, gallium,gold, indium, iridium, iron, lead, magnesium, mercury (as an amalgam),nickel, niobium, osmium, palladium, platinum, rhenium, rhodium,selenium, silicon (such as highly doped polycrystalline silicon),silver, tantalum, tin, titanium, tungsten, uranium, vanadium, zinc,zirconium, mixtures thereof, and alloys or metallic compounds of theseelements. Preferably, the metallic layer includes gold, platinum,palladium, iridium, or alloys of these metals, since such noble metalsand their alloys are unreactive in biological systems. The metalliclayer may be any thickness but preferably is 10 nm to 80 nm, morepreferably 20 nm to 50 nm.

[0081] In the laser ablation process, the metallic layer may be ablatedinto a pattern of micro-electrodes. The patterned layer may additionallybe coated or plated with additional metal layers. For example, themetallic layer may be copper, which is then ablated with a laser, intoan electrode pattern. The copper may be plated with a titanium/tungstenlayer, and then a gold layer, to form desired micro-electrodes.Preferably, however, in some embodiments, only a single layer of gold isused. One example of a useful metallic laminate is a polyester or otherflexible substrate such as a Kaladex film, coated with a layer of gold,preferably about 5 mils in thickness.

[0082] The conductive material is ablated with the laser to leave amicro-electrode array. Any laser system capable of ablation of theconductive material will be useful. Such laser systems are well knownand commercially available. Examples include excimer lasers, with apattern of ablation controlled by lenses, mirrors, or masks. A specificexample of such a system is the LPX-400, LPX-300, or LPX-200, both fromLPKF LASER ELECTRONIC, GMBH of Garbsen, Germany.

[0083] One specific example of a process for laser ablation is asfollows. Sheets of sensor traces are fabricated in a MicrolineLaser200-4 laser system (from LPKF). The system chamber includes a vacuumplaten atop of a LPKF-HS precision positioning X,Y table, laser mirrorsand optics, and a quartz/chromium photomask (International PhototoolCompany, Colorado Springs, Colo.) with the sensor components subdividedinto rectangular fields on the mask. Photomask positioning, X,Y tablemovement and laser energy are computer controlled. Sheets of metallaminate 22cm×22 cm in dimension are placed into the chamber onto thevacuum table. The table moves to the starting position and the Kr/Fexcimer laser (248 nm) is focused through the first field of thephotomask onto the metal laminate. Laser light passing through the clearareas of the photomask field ablates the metal from the metal laminate.Chromium coated areas of the photomask block the laser light and preventablation in those areas, resulting in a metallized sensor structure onthe laminate film surface. The complete structure of the sensor tracesmay require additional ablation steps through various fields on thephotomask.

[0084] Another method of preparing the described micro-electrode arrayis the use of flex circuit photolithography. Flex circuitphotolithography methods are well known. Two general methods offabricating flexible circuits include the “additive” method and the“subtractive” method. With the additive method, an IDA and associatedcircuitry can be built up on top of a non-conductive flexible substrate.With the subtractive method, a non-conductive flexible substrate can belaminated with a conductive material (e.g., a copper foil) andconductive material is patterned using conventional photolithographicand wet chemical etching techniques. Some conventional processing stepsinclude cleaning a substrate or intermediate; depositing conductivematerials onto a substrate, e.g., by vapor deposition,electrodeposition, or vacuum plasma sputtering; depositingnon-conductive or processing materials onto a substrate such as aphotoresist material; masking and developing a photoresist material in apattern defining an electrode; and removing excess developed ornon-developed materials such as photoresist materials or conductivematerials, to leave behind a desired arrangement of electricallyconductive and insulating materials.

[0085] According to one series of steps in flex circuitphotolithography, a substrate is prepared by cleaning, and a conductivematerial can be applied as a film to the substrate. Preferredthicknesses of a conductive layer (e.g., a gold conductive layer) can bein the range from about 500 to 1000 angstroms. It may be desirable toinclude a seed layer such as titanium or chromium between the conductivelayer and the substrate, to improve adhesion. A preferred conductivematerial can be gold, and a preferred method of application can besputtering, which has been found to provide very good adhesion.

[0086] A photoresist material can be applied to the conductive layer.Such photoresist materials are commercially known and available, withone example being Riston® CM206, from duPont. The thickness of thephotoresist can be chosen to advantageously affect the resolution of thefeature sizes of the electrode components. Improved resolution generallyprovides for better quality arrays, with fewer failures. There has beenfound a 1:1 relationship between the resolution of the smallest featuresize achievable, and the thickness of the dry film photoresist, withthinner photoresist films providing better resolution (a thickness ofabout 0.6 mils generally allows a feature spacing or width of 0.6 mils).Riston® CM206, in the form of a 0.6 mil thick roll of film, can be apreferred photoresist because it can be capable of resolving features,i.e., lines and spaces, on a lower micron scale, e.g., in the range of0.6 mils (15 microns) or lower. A photoresist layer often requiresheating. Riston® CM206 does not require prebaking. The material is a dryfilm photoresist and is applied to the gold substrate using a heatedlaminated roller system. Once laminated, the material is ready forprocessing (exposure to UV light, and development).

[0087] The laminated film can be cut to a convenient size, e.g., onefoot by one foot, and a pattern defining a micro-electrode array can becured or crosslinked. This can generally be accomplished by conventionalmethods, e.g., using a mask pattern and exposing the array pattern toultraviolet light, crosslinking the photoresist in the pattern of thearray. Unexposed, uncrosslinked, photoresist can be developed away usinga developing agent, which will typically be particular to thephotoresist composition (e.g., lithium carbonate is one developingagent; see the manufacturer's instructions). At the end of this step,the substrate will have an undisturbed layer of the conductive materialcoated thereon, with a photoresist design defining the pattern of thearray laid out on the conductive layer. This allows for unprotectedconductive material to be etched away using an etchant (e.g., KI/I₂), toproduce the IDA pattern in the conductive material. The remainingphotoresist can then be removed.

[0088] Once an array is prepared, e.g., by laser ablative methods, usinglaminated dry photoresist, spin coating, etching, or other techniques,further processing of the micro-electrode array can be used toincorporate the array into a useful electronic device such as abiosensor. Preferably, additional materials can be disposed onto thearray to form, for example, a spacer or insulating layer, optionallyincluding a well or a microchannel or capillary. A well refers to aspace over an array that defines the array. A microchannel or capillarymore specifically refers to a space or channel that is defined over thearray to allow the flow of a fluid over the array. The material used todefine the microchannel or capillary can be any of a variety ofmaterials useful insulating or spacing materials, sometimes referred toas “coverlay” materials, as well as other material useful for processingwith the described fabrication methods. An example is Pyralux coverlay,and similar materials would also be useful.

[0089] Methods useful to place a microchannel or capillary onto thearray include methods of mechanical lamination and mechanical removal ofmaterial to form a channel or capillary. One method would include afirst step of mechanically “punching” (e.g., die punching) the coverlaymaterial to cut away one or multiple portions of the material in theform of wells or channels, and then laminating the material to one or anumber of sensors such that the channel is present over the array.Another method includes those types of methods generally referred to as“kiss die cutting” or “kiss cutting,” which may be used to cut a well orchannel in a coverlay layer, and then the coverlay material may belaminated onto the subststrate with the well or channel over the array.One method of producing wells in a coverlay material is described, forexample, in U.S. patent application Ser. No. ______, entitled “Methodsto Fabricate Biomedical Devices with Wells and Micro-Environments andAssociated Products,” filed on even date herewith, and having attorneydocket No. 5051-552PR, the disclosure which is incorporated herein byreference.

[0090] A different example that includes a die punching method is asfollows. A spacer foil was prepared by coating an adhesive, FastbondTM30-NF Contact Adhesive to a wet thickness of 25 μm onto a 5 milpolyester film such as that sold under the trademark Melinex® S (DuPontPolyester films, Wilmington Del.) using a wire bar coater from ThomasScientific of Swedesboro, N.J. The coated top foil was dried for 2minutes at 50C in a horizontal air flow oven. The dried adhesive on thesheet was covered with either silicon or teflon release liner. Capillarychannels and electrode contact well patterns were kiss cut into thesheet using an Aristomat 1310 ditigal die cutting system (Aristo GraphicSysteme GmbH & Co., Hambrug Germany). The spacer sheet can then beregistered and laminated to an ablated sheet of sensor traces, asdescribed above. Channels and electrode contact wells can also beproduced using die punching processes in a similar fashion.

[0091] Another specific method by which to dispose a capillary ormicrochannel onto a micro-electrode array would be by methods of flexcircuit photolithography. Accordingly, a photoimageable coverlaymaterial such as Vacrel 8140®, (a dry film coverlay can be preferred)can be vacuum laminated onto the gold/plastic laminate. Multiple layersof various chosen thicknesses can be added to control the depth of thecapillary chamber (see infra). The sheet can be exposed to ultravioletlight through a mask pattern to define capillary chambers. The exposedlaminated sheet is developed by conventional methods, e.g., using 1%K₂CO₃, to remove crosslinked photopolymer coverlay material and leavebehind components of a capillary. The sheet is generally thereaftercured, e.g., at 160C. for 1 hour.

[0092] In fabricating the capillary, the depth of the chamber can becontrolled by choosing the coverlay material or materials used,according to thickness. Vacrel 8140® film has a thickness of 2, 3, or 4mil (100 μm). Pyralux PC® 1000, 1500, have 2000 have maximum thicknessesof 2.5 mils (63.5 μm), so double layer lamination gives a chamber depthof 127 μm. Pyralux 1010 has a thickness of 1 mil or 25.4 μm. Capillarieswith depths of greater than or equal to 100 μm have been found to allowfast fill of blood with hematocrits from 20 to 70% to reliably flow intothe chamber. Capillary depths of less than 100 microns to 25 microns canbe used for other biological fluids such as serum, plasma, intersticialfluid, and the like.

[0093] A chemical coating may also be disposed onto the array. First,however, it may be beneficial to clean the sensors. By one cleaningmethod, a sheet of sensors as described can be plasma cleaned in aBranson/IPC Plasma Cleaner according to steps such as the following: (1)O₂ for 1 minute at 800 watts; (2) O₂/Argon(Ar) (70/30) for 3 minutes at220 watts; (3) Ar for 2 minutes at 150 watts.

[0094] A chemical coating, as described, may be dispensed onto thearray, e.g., into each capillary chamber and over the interdigitatedarrays, by known methods. The method of dispensing is preferably capableof reproducibly and consistently delivering very small volumes of achemical composition, onto the array—e.g., volumes in the range ofhundreds of nanoliters, e.g., 625 nanoliters. As an example, such acoating may be dispensed using known syringe and metering techniques andapparatuses, including dispensing systems sold under the trade nameMicrodot (from Astro Dispense Systems, a DCI Company of Franklin, Mass.02038-9908) and systems sold by BioDot Inc., Irvine, Calif. The coatingsmay then be dried of solvent. The inlet ports are opened, and a top foilcoated with a hydrophilic adhesive is applied over the capillary chamberusing heat and pressure to form the completed three-dimensional sensorstructure.

[0095] The top foil can be any continuous film capable of defining oneside of the capillary, and preferably being capable of appropriateprocessing, e.g., as described herein. Exemplary materials for the foilcan include plastic films such as polyethylene naphthalate (PEN), filmtype Kadalex 1000, 7 mil thick.

[0096] Any of a variety of hydrophilic adhesives can be used to bond thetop foil to the sensor. Two part thermoset adhesives such aspolyurethane mixtures and isocyanate mixtures can be used, e.g., 38-8668(polyurethane) and 38-8569 (isocyanate) (95:5 wt./wt.) from NationalStarch and Chemical Co. of Bridgwater N.J., or, a two part epoxy systemssuch as that sold under the trademark Scotch Weld™ 2216 B/A (3M AdhesiveDiv., St. Paul Minn.), as well as contact adhesives such as that soldunder the trademark Fastbond™ 30-NF Contact Adhesive, provided that theyexhibit acceptable sealing properties to the crosslinked coverlaysurface. A preferred adhesive was found to be a mixture of Fastbond™30-NF Contact Adhesive and the surfactant Triton™ X-100 (Union Carbide,Danbury Conn.), 93%:7% wt./wt.

EXAMPLES

[0097] The following describes a process useful for preparing a sensoraccording to the invention, comprising an interdigitated array disposedon a flexible substrate. According to the method, a gold film can bedeposited onto 7 mil thick Kaladex® film using a planar DC magnetronsputtering process and equipment, from Techni-Met Inc., Windsor, Conn.The thickness of the gold film can range from 30 to 200 nm, with apreferred thickness being 100 nm. Seed layers of chromium or titaniumcan be sputtered between the plastic film and the gold to promote betteradhesion of the gold to the plastic substrate, however, gold layerssputtered directly onto the plastic film can exhibit sufficientadhesion.

[0098] The interdigitated array and connectors can be fabricated usingbatch photolithography processes common to the flex circuit industry.Electrodes with combinations of finger width and spacing between fingersin the range of 21 to 50 um were easily fabricated using theseprocesses. A preferred configuration of the array was 21 total fingers(10 working electrode fingers and 11 counter electrode fingers), withfinger dimensions of 25 microns (width) by 1 millimeter (length), with21 micron spacing between the fingers.

[0099] After the gold was applied to the flexible substrate, a dry filmphotopolymer resist was laminated to the gold/plastic film. A dry filmresist such as that sold under the trademark Riston® CM206 (duPont) waspreferred. The Riston® CM206 photoresist was first wet laminated ontothe gold surface of 12″×12″ gold/plastic panels using a HRL-24 hot rolllaminator (from duPont). The sealing temperature and lamination speedwere 105° C. and 1 meter per minute. The laminated panel was placed in aTamarack model 152R exposure system, from Tamarack Scientific Co., Inc.,Anaheim, Calif. The release liner was removed from the top surface ofthe photoresist. A glass/emulsion photomask of the IDA configuration wasproduced by Advance Reproductions Corporation, North Andover, Mass. Theemulsion side of the mask was treated with an antistick coating(Tribofilm Research Inc., Raleigh, N.C.), and was placed directly ontothe photoresist surface of the panel. The laminated panel was exposed toultraviolet light of 365 nm through the photomask using an exposureenergy of 60 mJ/cm². Exposed photoresist was stripped from the panel ina rotary vertical lab processor (VLP-20), Circuit Chemistry Equipment,Golden Valley, Minn., using 1% potassium carbonate, at room temperature,for 30 seconds using a nozzle pressure of 34 psi. Exposed gold on thesheet was then stripped using an etch bath containing a solution of: 4parts I₂:1 part KI:40 parts water vol./vol.; and 0.04 gram Fluorad™fluorochemical surfactant FC99, (3M, St. Paul, Minn.) per 100 gramsolution, added to the bath to ensure wetting of the photoresist. Airwas bubbled through the bath during the etch process to obtain uniformagitation of the bath mixture. The panel was rinsed with deionized waterand residual Riston® CM206 was removed in a 3% KOH bath.

[0100] Sensor chambers were fabricated using dry film photoimageablecoverlay materials such as that sold under the trademark Vacrel® 8140(duPont) or Pyralux® PC series (duPont). The chamber dimensions can beaccurately defined by flex circuit photolithography. Depth of thechamber was controlled by the thickness of the coverlay materials used,and whether single or multiple layers of the coverlay dry film wereused. A preferred chamber depth was 125 microns (5 mil). This chamberdepth was achieved by sequential lamination of different coverlaymaterials as follows: three mil thick Vacrel® 8130 was first laminatedto the electrode side of the substrate using a HRL-24 (duPont) heatedroll laminator at room temperature, using a roller speed of 1 meter perminute. The electrode panel was then vacuum laminated in a DVL-24 vacuumlaminator (duPont) using settings of 120° F., 30 second vacuum dwell,and a 4 second pressure to remove entrapped air between the coverlayfilm and the electrode substrate. Two mil thick Vacrel 8120 waslaminated next to the Vacrel® 8130 surface using the HRL-24 at roomtemperature, with a roller speed of 1 meter/min. The panel was thenvacuum laminated again in the DVL-24 vacuum laminator using a 30 secondvacuum dwell, 4 second pressure, to remove entrapped air between the twocoverlay films.

[0101] The laminated electrode sheet was placed in the Tamarack 152Rsystem and was exposed to ultraviolet light at 365 nm through thephotomask for 22 seconds using an exposure intensity of 17 mW/cm². Theartwork for the capillary chamber was a 1 millimeter by 4 millimeterrectangle centered over the electrode finger array and starting 1millimeter below the fingers. The exposed coverlay was stripped from thepanel to reveal the sensor chamber rectangle using the VLP-20 CircuitChemistry Equipment) in 1% K₂C0₃, at 140° F., for 75 seconds using anozzle pressure of 34 psi. The developed laminate structure was rinsedin deionized water, and then cured at 160° C. for 1 hour to thermallycrosslink the coverlay material. This completed the construction of thesensor base.

[0102] The panel of the base sensors was plasma cleaned to removeresidual photoresist and coverlay material from the exposed gold surfaceof the interdigitated array structure. The panel was placed in a barreletcher, a Barnstead/IPC model P2100 from Metroline/IPC of Corona, Calif.The panel was first exposed to an oxygen plasma for 1 minute at 800watts and 1.1 torr pressure to oxidize the panel surface. It was thenetched in an oxygen/argon plasma mixture (70/30 vol./vol.) for 3minutes, at 225 watts and 1.5 torr pressure, and was finally stripped inan argon plasma for 2 minutes, at 150 watts and 2 torr pressure.

[0103] The chemical coating was formulated for measurement of d-glucosein a human blood sample. The chemical coating was reactive with thesample in a manner effective to generate an electrical output signalindicative of the level of glucose in the sample. The coating included amediator, enzymes, and a cofactor. The coating further comprised filmforming agents and detergents conferring durability and providinghydrophilicity. The ingredients are listed in Table 1; unless statedotherwise, all concentrations refer to the concentration of a givensubstance in a wet-coating, prior to the deposition and drying of thecoating onto the array.

[0104] The chemical coating was formulated from several sub-mixtures ofcomponents. A first mixture contained glycerophosphate buffer, from ICNBiomedicals Inc. Aurora, Ohio; Medium Viscosity Alginic acid, from SigmaChemical Co., St. Louis, Mo.; Natrosol 250M, from Hercules Inc.,Wilmington, Del.; and Triton® X-100, from Union Carbide, Danbury Conn.These components were added to a volume of distilled water sufficient tomake a 250 gram solution of the buffer/polymer/surfactant (see Table 1).The solution was mixed overnight to allow complete hydration of theNatrosol and Alginic acid. The pH of the completed solution was adjustedto 6.9 to 7.0 with concentrated hydrochloric acid. This solution isknown hereinafter as “Solution A.”

[0105] A second solution prepared was a concentrated enzyme/cofactormatrix. 8.2 milligrams pyrrolo-quinoline-quinone (PQQ), Fluka,Milwaukee, Wis., was added to 25.85 grams of Solution A. The resultingmixture was sonicated until the PQQ was completely in solution. 1.1394grams of the enzyme, Glucose-De-oxidoreductase (GlucDor), from RocheMolecular Biochemicals, Indianapolis, Ind., was added to the solution.The final mixture was rocked for 2 hours to allow formation of theGlucDor/PQQ holoenzyme. The completed solution will be referred to as“Solution B.”

[0106] Potassium ferricyanide was added to the composition as follows:4.4173 grams of potassium ferricyanide, from J. T. Baker, Phillipsburg,N.J., was added to 70.58 grams of Solution A. The resulting solution wasmixed until the ferricyanide was completely in solution. The completedsolution will be referred to as “Solution C.”

[0107] The final coating composition was completed by combining 63 gramsof Solution C to 25 grams Solution B. This composition was rocked in thedark for 1 hour to thoroughly mix. TABLE 1 Formulation per 100 grams ofcoating Concentration/ Wet mass Component activity (g) Dry mass/sensor(mg) Distilled Water 88.487 Disodium   150 mM 4.359  0.0287Glycerophosphate pH 6.98 Trehalose    1% wt/wt 1.000  0.0066 Natrosol 0.3% wt/wt 0.300  0.002 Alginic acid  0.4% wt/wt 0.400  0.0026 Mediumviscosity Triton X-100 0.025% wt/wt 0.025  0.00016 Pyrrolo-quinoline-0.261 mM 0.0082  5.3382 × .10 − 5 Quinone (PQQ) GlucDor Enzyme  2034u/mg 1.1394  0.0075 15.23 (units) Potassium   137 mM 4.2814  0.0281Ferricyanide

[0108] A preferred method for applying the chemistry matrix to thesensor chamber (IDA) is a discrete dispense of 500 nanoliters of thecoating solution into the 1 millimeter×4 millimeter chamber using amicrodispensing system such as that sold under the trademark of BioJetQuanti3000™, BioDot Inc., Irvine, Calif. The coating covered both theworking and counter electrodes of the IDA. The coating was dried for 1.5minutes at 45° C. in a horizontal air flow oven, VWR ScientificProducts, Chicago Ill.

[0109] The hydrophilic top foil was prepared by coating an adhesivemixture (e.g., a mixture of Fastbond™ 30-NF Contact Adhesive and thesurfactant Triton™ X-100 (Union Carbide, Danbury Conn.), 93%:7% wt/wt.)to a wet thickness of 25 u onto 5 mil polyester film such as that soldunder the trademark Melinex® “S” (duPont Polyester Films, WilmingtonDel.) using a wire bar coater from Thomas Scientific, Swedesboro, N.J.The coated top foil was dried for 2 minutes at 50° C. in a horizontalair flow oven (VWR Scientific Products). The capillary chamber wasopened by cutting 1 millimeter in from the front edge of the capillarychamber with a pair of scissors. The dried coated top foil was appliedto the sensor, allowing approximately a 0.5 mm space between the backedge of the chamber and the edge of the top foil as an air vent. The topfoil was sealed to the sensor surface using a 5 ton press with a heatedtop platen, at 81° C., 60 psi for 5 seconds. The panel of completedsensors was cut into individual sensors and stored desiccated at 8% RHuntil tested.

[0110] The sensors were evaluated using chronoamperometryelectrochemical techniques on test stands such as that sold under thetrademark of BAS™ 100W Electrochemical Workstation, BioanalyticalSystems, Inc. West Lafayette, Ind. The preferred electrochemical teststand used in the evaluation of the electrodes was a dedicated teststand for DC chronoamperometric current measurement for assay potentialsfrom ±1 volt.

[0111] The sensors may be used to determine the concentration of ananalyte, such as glucose, in a fluid sample by performing the followingsteps:

[0112] Set up the test stand parameters:

[0113] In accordance with a “drop detect” system, an initial potentialdifference is established between the working and counter electrodes—300mV (millivolts)—to start timing of the analysis sequence. Currentresponse to this potential is triggered by contact of the array with afluid sample.

[0114] The initial current response upon application of the testsolution to the sensor chamber is generally greater than 0.4 microamps.

[0115] The time (delay period) between the threshold trigger andre-application of the 300 mV potential difference (assay potential) isgenerally 3 seconds.

[0116] The assay period, after re-application of the 300 mV potentialdifference between the working and counter electrodes of the sensor isgenerally 9 seconds.

[0117] In more detail:

[0118] Insert the sensor into the test stand connection. Applyapproximately 0.3 uL of a fluid sample to the opening of the capillarychamber. Fluid will flow into the chamber by capillary action coveringthe chemical coating applied to the working and counter electrodes. Thethreshold current will be triggered when the sample fluid covers thenearest working and counter electrode fingers. Once triggered, thepotential difference will go to open circuit for a 3 seconds, during thedelay period.

[0119] During the delay period, reaction will occur between thereactants (analyte, enzyme/cofactor, and the oxidized form of themediator), resulting in reduction of the mediator.

[0120] The 300 mV assay potential difference is re-applied between theelectrodes after the 3 second delay. This causes electro-oxidation ofthe reduced mediator at the surface of the working electrode.

[0121] The current/time reaction profiles of the assay show acharacteristic pseudo-steady-state current/time plateau starting 0.5 to1.5 seconds after re-application of the 300 mV assay potential to thesensor. Currents at fixed assay period points chosen in this plateauregion were proportional to the concentration of analyte in the samplefluid. Assay endpoints were chosen in such a manner give a linear doseresponse for glucose concentrations from 0 to 600 mg/dL. See FIG. 7.

1. A disposable electrochemical sensor for the detection of an analytein a liquid sample comprising a working electrode and a referenceelectrode disposed within a sample-receiving cavity, a reagent layerdisposed within the sample-receiving cavity over at least the workingelectrode, said reagent layer comprising an enzyme for producing anelectrochemical signal in the presence of the analyte, wherein thesample-receiving cavity has a volume of less that 1.0 μl and wherein thesensor provides an accurate reading of the amount of analyte in a periodof 10 seconds or less.
 2. The sensor of claim 1, wherein the reagentlayer further comprises an electron transfer mediator.
 3. The sensor ofclaim 2 in which the mediator is selected from the group consisting offerricyanide, osmium (III)-(bipyridyl)-2-imidazolyl-chloride, Meldolablue, [Ru(NH₃)₅pyz]₂(SO₄)₃, and nitrosoanaline derivatives.
 4. Thesensor of claim 3, wherein the mediator is nitrosoanaline derivatives.5. The sensor of claim 3, wherein the mediator is ferricyanide.
 6. Thesensor of claim 2 in which the analyte is selected from the groupconsisting of glucose, cholesterol, HDL cholesterol, triglycerides,lactate, lactate dehydrogenase, pyruvate, alcohol, uric acid, and3-Hydroxybutyric acid
 7. The sensor of claim 6, wherein the analyte isglucose.
 8. The sensor of claim 7 in which the mediator is selected fromthe group consisting of ferricyanide, osmium(III)-(bipyridyl)-2-imidazolyl-chloride, Meldola blue,[Ru(NH₃)₅pyz]₂(SO₄)₃, and nitrosoanaline derivatives.
 9. The sensor ofclaim 8, wherein the mediator is ferricyanide.
 10. The sensor of claim 7in which the enzyme is selected from the group consisting of glucoseoxidase, glucose dehydrogenase and diaphorase.
 11. The sensor of claim10 in which the enzyme is glucose dehydrogenase.
 12. The sensor of claim10 in which the enzyme is glucose oxidase.
 13. The sensor of claim 10 inwhich the mediator is selected from the group consisting offerricyanide, osmium (III)-(bipyridyl)-2-imidazolyl-chloride, Meldolablue, [Ru(NH₃)₅pyz]₂(SO₄)₃, and nitrosoanaline derivatives.
 14. Thesensor of claim 13, wherein the mediator is ferricyanide.
 15. The sensorof claim 1, wherein the reagent layer is disposed over both the workingelectrode and the reference electrode.
 16. The sensor of claim 1,wherein both the working electrode and the reference electrode areconductive carbon electrodes.
 17. A method of determining theconcentration of an analyte in a liquid sample comprising: providing atest sensor including a sample receiving cavity having a volume of lessthan 1 μl, the sensor including working and counter electrodes disposedwithin the sample receiving cavity, the sensor further including areagent layer comprising an enzyme and a mediator, the reagent layerbeing disposed on at least the working electrode, the enzyme andmediator being selected to react with the analyte to generate anelectrochemical signal representative of the concentration of theanalyte in the liquid sample; admitting the liquid sample into thesample receiving cavity; detecting the time at which the liquid sampleenters the sample cavity; following said detecting, controlling thevoltage or current across the working and counter electrodes; and withinten seconds of said detecting, obtaining a readout of the concentrationof the analyte in the liquid sample.
 18. The method of claim 17 in whichsaid obtaining comprises measuring the electrochemical signal receivedfrom the working electrode and determining the concentration of theanalyte from the electrochemical signal.
 19. The method of claim 17 inwhich said controlling comprises applying a DC voltage of 100-500 mVacross the working and counter electrodes, said obtaining comprisingmeasuring the current received from the working electrode anddetermining the concentration of the analyte from the measured current.20. A disposable electrochemical sensor for the detection of an analytein a liquid sample comprising a working electrode and a referenceelectrode disposed within a sample-receiving cavity, a reagent layerdisposed within the sample-receiving cavity over at least the workingelectrode, said reagent layer comprising an enzyme for producing anelectrochemical signal in the presence of the analyte, wherein thesample-receiving cavity has a volume of less than about 0.4 μl, andwherein the sensor provides an accurate reading of the amount of analytein a period of 10 seconds or less.
 21. The sensor of claim 20 in whichthe sample receiving cavity has a volume of about 0.1 μl to about 0.4μl.
 22. The sensor of claim 21 in which the sample receiving cavity hasa volume of about 0.1 to about 0.25 μl.
 23. The sensor of claim 20 inwhich the sample receiving cavity has a height of about 25 to about 200μm.
 24. The sensor of claim 20 in which the sample-receiving cavity hasa width of about 1 mm.
 25. The sensor of claim 20 in which thesample-receiving cavity has a length of about 4 mm.
 26. The sensor ofclaim 20 in which the gap between the working and reference electrodesis 15 μm to 50 μm.
 27. The sensor of claim 20 in which each of theworking and reference electrodes has a thickness of 10 μm to 200 μm. 30.A method for detecting the concentration of an analyte in a liquidsample comprising: providing a disposable electrochemical sensorincluding a working electrode and a reference electrode disposed withina sample-receiving cavity, a reagent layer disposed within thesample-receiving cavity over at least the working electrode, the reagentlayer comprising an enzyme for producing an electrochemical signal inthe presence of the analyte, the sample-receiving cavity having a volumeof less than about 1 μl; admitting the liquid sample into the samplechamber; detecting the application time at which the liquid sampleenters the sample chamber; following said application time, applying avoltage across the working and reference electrodes; and within tenseconds of said application time, obtaining a readout of theconcentration of the analyte in the liquid sample.
 31. The method ofclaim 30 in which the sample-receiving cavity has a volume of less thanabout 0.4 μl.
 32. The sensor of claim 31 in which the sample receivingcavity has a volume of about 0.1 μl to about 0.4 μl.
 33. The sensor ofclaim 32 in which the sample receiving cavity has a volume of about 0.1to about 0.25 μl.
 34. The sensor of claim 30 in which the samplereceiving cavity has a height of about 25 to about 200 μm.
 35. Themethod of claim 30 which includes incubating the liquid sample for anincubation time extending from the application time to said applying avoltage.
 36. The method of claim 35 in which the incubation time is fromabout 0.5 seconds to about 6 seconds.
 37. The method of claim 36 inwhich the incubation time is about 2 seconds to about 6 seconds.
 38. Themethod of claim 37 in which the incubation time is about 3 seconds. 39.The method of claim 36 in which the incubation time is about 0.5seconds.
 40. The method of claim 30 in which said obtaining a readoutcomprises obtaining the readout in a test time from about 0.5 to about 2seconds after said applying a voltage.
 41. The method of claim 40 inwhich the test time is 0.5 to 1.5 seconds.
 42. The method of claim 30 inwhich said obtaining a readout comprises obtaining the readout less thanabout 5 seconds after the application time.
 43. The method of claim 42in which said obtaining a readout comprises obtaining the readout about4 seconds after the application time.
 44. The method of claim 30 inwhich said applying a voltage comprises applying a voltage of about 100mV to about 500 mV.
 45. The method of claim 44 in which said applying avoltage comprises applying a voltage of about 300 mV.
 46. A disposableelectrochemical sensor for the detection of an analyte in a liquidsample comprising a working electrode and a reference electrode disposedwithin a sample-receiving cavity, a reagent layer disposed within thesample-receiving cavity over at least the working electrode, saidreagent layer comprising an enzyme for producing an electrochemicalsignal in the presence of the analyte, wherein the sample-receivingcavity has a height of about 25 μm to about 200 μm, and wherein thesensor provides an accurate reading of the amount of analyte in a periodof 10 seconds or less.
 47. The sensor of claim 46 in which the samplereceiving cavity has a height of about 25 μm to about 100 μm.
 48. Thesensor of claim 46 in which the sample receiving cavity has a height ofat least about 100 μm.
 49. The sensor of claim 48 in which the samplereceiving cavity has a height of about 125 μm.
 50. The sensor of claim46 in which the sample receiving cavity has a volume of less than about1 μl.
 51. The sensor of claim 50 in which the sample receiving cavityhas a volume of about 0.1 to about 0.4 μl.
 52. A method for determiningthe concentration of an analyte in a liquid sample comprising providinga disposable electrochemical sensor comprising a working electrode and areference electrode disposed within a sample-receiving cavity, a reagentlayer disposed within the sample-receiving cavity over at least theworking electrode, the reagent layer comprising an enzyme for producingan electrochemical signal in the presence of the analyte; admitting theliquid sample into the sample receiving cavity; detecting theapplication time at which the liquid sample enters the sample chamber;following said application time, applying a voltage across the workingand reference electrodes for a time of less than about 2 seconds; andwithin ten seconds of said application time, obtaining a readout of theconcentration of the analyte in the liquid sample.
 53. The method ofclaim 52 in which said applying a voltage comprises applying a voltagefor a time from about 0.5 to about 2 seconds.